The diverse functions of the nervous system, which range from sensory perception and motor coordination to motivation and memory, depend on precise connections formed between distinct types of nerve cells. The development of this complex system occurs in several steps. In vertebrates, first, a uniform population of neural progenitor cells called neural plate cells are recruited from the sheet of ectodermal cells that have not yet committed to a specific pathway of differentiation. Once recruited, neural plate cells rapidly begin to differentiate, acquiring new properties that characterize the cells as immature neurons and glial cells. The first signaling event that allows a single cell type, that is neural plate cells, to give rise to a large number of neuronal and cell types depends on multiple biochemical factors and processes (see, for example, Principles in Neural Science (4th), eds. Kandel, Schwartz and Jessell, Elsevier Science Publishing Company: N.Y., 2000).
The differentiation of the neural plate from uncommitted ectoderm depends on signals secreted by a group of cells called the organizer region. Cells from the organizer region produce factors that induce and suppress development of neural tissues. Mesodermal cells in the organizer region and in the notochord provide an inductive signal that is mediated by a protein called sonic hedgehog. Sonic hedgehog is a member of a family of proteins related to the gene hedgehog (hh). This single protein, acting through short range and long range signaling activities can induce the differentiation of several mature neuronal types: floor plate cells, motor neurons and ventral interneurons.
The hedgehog gene was first identified and isolated in Drosophila where its multiple roles include patterning of larval segments and adult appendages. Vertebrate hh homologues also are involved in many aspects of developmental patterning. Hedgehog protein biogenesis has been best studied for the Drosophila protein but very likely is similar for Hedgehog proteins from all species. After cleavage of an amino-terminal signal sequence on entry into the secretory pathway, the Hh protein undergoes an intramolecular autoprocessing reaction that involves internal cleavage between the Gly-Cys residues of an absolutely conserved Gly-Cys-Phe (GCF) tripeptide. The amino-terminal product of this cleavage, which is the species active in signaling, also receives a covalent cholesteryl adduct. Autoprocessing at this site and covalent linkage to cholesterol have been experimentally confirmed for the Shh protein.
In Drosophila, a hedgehog protein from a construct truncated at the internal site of cleavage is active in signaling, but this protein is not spatially restricted in its signaling activity and therefore causes gross mispatterning and lethality in embryos. The autoprocessing reaction thus is required not only to release the active signal from the precursor but also to specify the appropriate spatial distribution of this signal within developing tissues, presumably through insertion of the cholesteryl moiety into the lipid bilayer of the plasma membrane. Recent studies also have revealed palmitoylation of the amino-terminal cysteine of the amino-terminal signaling domain of the Shh secreted protein (Shh-N); the occurrence of this second lipid modification is regulated by autoprocessing and may also influence the activity and distribution of Shh-N.
Several components have been identified as candidates for receptor function in transduction of the hh protein signal. The patched (ptc) gene, originally identified in Drosophila, encodes a multipass transmembrane protein, a patched receptor (Ptc). ptc mutations in Drosophila embryos cause inappropriate activation of wingless gene expression, a phenotype opposite that of hh mutations, thus suggesting that ptc functions as a negative effector in hh signaling. The observations that hh ptc double mutant embryos resemble ptc mutants and that, in a ptc mutant background, ectopic Hh expression produces no further phenotypic effects, together suggest that the Ptc gene product acts downstream of Hh to regulate its signaling activity. Genetic epistasis studies further suggest that the smoothened gene (smo), which encodes another transmembrane protein (Smo), functions downstream of ptc in the hh signaling cascade. Because smo is required for hh signaling, it has been proposed that Smo activates the Hh pathway and that Ptc inhibits Smo activity. Genetic mosaic analysis in the Drosophila wing imaginal disc showed that Ptc has, in addition to a cell-autonomous negative effect on Hh signaling, an ability to sequester the Hh protein and prevent its movement to adjacent cells.
Vertebrate homologues of both ptc and smo genes have been identified. Shh-N was found to bind to cells expressing Ptc or both Ptc and Smo, but not to cells expressing Smo alone. Moreover, Ptc interacted with Smo independently of the presence of Shh-N, suggesting that the two transmembrane proteins form a complex. An integrated view of Drosophila genetic analyses and biochemical studies of vertebrate homologues suggests a model in which the Ptc-Smo complex might function as Hh receptor, with direct binding of Hh to Ptc releasing Smo activity from inhibition by Ptc. It must be noted, however, that these biochemical studies did not examine the role of a physical interaction between Shh-N and Ptc in activation of the Shh pathway. In addition, these biochemical studies did not exclude the possibility that Shh-N interacts not directly with Ptc but with another component of a complex that includes Ptc, because the crosslinked binding complexes were extremely large and were not analyzed with regard to their composition.
The model just described assumes a role for Shh-N as a ligand for a receptor. The crystal structure of the Shh-N protein, however, suggested an alternative possibility. This structure revealed a zinc ion coordinated in an arrangement remarkably similar to that of thermolysin, carboxypeptidase A, and other zinc hydrolases. Even more striking is the remarkable similarity in folded structure of a portion of Shh-N to the catalytic domain of D,D-carboxypeptidase from Streptomyces albus, a cell wall enzyme closely related in structure and activity to other bacterial enzymes involved in conferring vancomycin resistance. Although the functional role of this putative hydrolase in Shh-N is not known, one possibility is that signaling requires Shh-N hydrolytic activity on as yet unknown substrates. Thus, several fundamental questions about the mechanisms of Shh-N signaling remain unanswered. To illuminate these issues, the present invention provides Shh-N mutations that abolish zinc hydrolase activity within Shh-N and Shh-N proteins with alterations in evolutionarily conserved surface residues.